A variety of ionization sources, for the analysis of molecules with medium-high molecular weight (like peptides and proteins) are essential components of modern mass spectrometric instruments. The ionization source transforms neutral molecules into ions which can be analyzed by mass spectrometry.
A mass spectrometer generally has the following components:
(1) a device, usually a Liquid Chromatograph, for the separation or de-salting of the molecules contained in a sample;
(2) an ionization source, contained in a chamber, to produce ions from the analyte;
(3) at least one analyzer or filter which separates the ions according to their mass-to-charge ratio;
(4) a detector that counts the number of the ions;
(5) a data processing system that calculates and plots a mass spectrum of the analyte.
The mass spectrometry techniques currently used for the analysis of macromolecules and, especially, proteins and peptides are based on the Electrospray Ionization (ESI) (U.S. Pat. No. 5,756,994; Cunsolo V, Foti S, La Rosa C, Saletti R, Canters G W, Verbeet M. Ph. Rapid Commun. Mass Spectrom. 2001; 15: 1817; Wall D B, Kachman M T, Gong S S, Parus S J, Long M W, Lubman D M. Rapid Commun. Mass Spectrom. 2001; 15: 1649; Fierens C, Stöckl D, Thienpont L M, De Leenheer A P. Rapid Commun. Mass Spectrom. 2001; 15: 1433; Li W, Hendrickson C L, Emmett M R, Marshall A G. Anal. Chem. 1999; 71: 4397; Fierens C, Stöckl D, Thienpont L M, De Leenheer A P. Rapid Commun. Mass Spectrom. 2001; 15: 451) and Matrix Assisted Laser Desorption Ionization (MALDI) (U.S. Pat. No. 5,965,884; Cozzolino R, Giorni S, Fisichella S, Garozzo D, La fiandra D, Palermo A. Rapid Commun. Mass Spectrom. 2001; 15: 1129; Madonna A J, Basile F, Furlong Ed, Voorhees K J. Rapid Commun. Mass Spectrom. 2001; 15: 1068; Basile A, Ferranti P, Pocsfalvi G, Mamone G, Miraglia N, Caira S, Ambrosi L, Soleo L, Cannolo N, Malorni A. Rapid Commun. Mass Spectrom. 2001; 15: 527; Galvani M, Hamdan M, Rigetti P G. Rapid Commun. Mass Spectrom. 2001; 15: 258; Ogorzalek Loo R R, Cavalcali J D, VanBogelen R A, Mitchell C, Loo J A, Moldover B, Andrews P C. Anal. Chem. 2001; 73: 4063).
Both techniques are highly effective for the production of ions of biomolecules in the gas phase, to be subsequently analyzed by Mass Spectrometry (MS).
In the case of ESI, multicharge ions of medium/high molecular weight compounds are produced. The mass of macromolecule compounds is then obtained using specific software algorithms.
Mass spectrometry represents an essential technology in the analytical field. It is usually coupled with other separative techniques, so as to identify chemical compounds and quantify complex biological mixtures. Proteins, for instance, are first separated, collected and then digested with Trypsin. The masses of the resulting peptides are determined by mass spectrometry (normal scan MS or tandem mass spectrometry MS/MS). In the case of the MS/MS approach, peptide ions of a single m/z ratio are fragmented by collision induced dissociation (CID) and then analyzed using various mass analyzers (triple quadrupole, ion trap, Fourier transform-ion cyclotron resonance). Each peptide gives origin to specific mass patterns for a given amino acid sequence. The peptide sequences can be obtained by computer analysis of the data using a dedicated software (database search and de novo sequence software). In order to obtain good MS/MS spectra doubly charge peptide ions are preferably fragmented (Cramer R, Corless S. Rapid Commun. Mass Spectrom. 2001; 15: 2058). The electrospray and MALDI techniques when are applied to the analysis of peptides with high molecular weight (2000-4000 Thompson (Th)) using the MS/MS approach have some limitations. For instance, when proteins or peptides with high molecular weight are analyzed, ESI multicharge ions are produced. These ions give rise to complex fragmentation spectra, difficult to interpret. For this reason only peptides with a maximum of 15 amino acidic residues can be analyzed by tandem mass spectrometry. In the case of MALDI only mono-charge ions are usually obtained. If the MALDI source is coupled with Time of Flight Mass Analyzer (TOF) the technique used to fragment the ions is the post source decay (PSD). This fragmentation technique give rise to some additional problems; in order to obtain good fragmentation spectra it is usually necessary to use peptide derivatization. A MALDI atmospheric pressure source has recently been coupled with an ion trap analyzer. This configuration makes possible the structural analysis of peptides by MS/MS and MS3. However, it must be emphasized that the MALDI source produces, mainly, mono-charge peptide ions that produce fragmentation spectra more complex and less specific than those obtained by fragmentation of the bi-charge ions.
Another problem that affect both MALDI and ESI techniques is represented by the decrease in sensitivity when salts are present in the sample. In the case of ESI the problem may be solved by coupling the mass spectrometer with a pre-analytical separation step, such as by the use of an High Performance Liquid Chromatographer (HPLC) or other de-salting techniques. This obviously introduces another step in the whole procedure of analysis. The HPLC technique on the other hand cannot be used for the case of MALDI because in this case it is necessary to co-crystallize the analyte with a matrix molecule. Salts contained in the sample must, however, be eliminated before of the crystallization step by well known additional treatments of the sample.